Abstract:
The cornea, a transparent ‘window’ of the eye, is composed of three distinct layers. Among
these, keratocytes in the corneal stroma help maintain transparency, heal wounds, and
synthesize stromal components. In Keratoconus, these cells are progressively lost such that the
lamellar structure that maintains transparency is distorted, leading to vision loss. In serious
instances, corneal transplants are performed; however, a global lack of donor tissue urges the
need to develop alternative therapeutic strategies. The umbilical cord is an abundant source of
mesenchymal stem cells (MSCs), which have the potential to differentiate into functional adult
keratocytes. Yet, there are varying isolation and differentiation success rates. This research
aimed to optimise the isolation of MSCs by exploring the characteristics and distribution of
MSCs in the umbilical cord, and establishing the location with the largest number of MSCs.
The structural and cellular anatomy of the umbilical cord in three dimensions were explored in
umbilical cords by H&E and immunohistochemistry (IHC). A novel protocol for identifying
MSCs in situ by IHC triple labelling of MSC markers was developed. Antibody validation was
performed by Western Blot. Expression of these MSC markers were measured using droplet
digital polymerase chain reaction. Cell distribution was quantified by DAPI staining.
Vimentin, laminin, and αSMA were expressed by all the cells across cross-sections of
sequential regions along the umbilical cord, confirming their mesenchymal origin, presence of
basement membranes and myofibroblastic phenotypes. Cytokeratin was localised to the
epithelial cord lining. All stromal cells in the umbilical cord displayed expression of CD105,
CD90, and CD73, the three acclaimed positive MSC markers. CD34 and CD45, negative MSC
markers, were also expressed. Cells furthest from the placental insertional region of the
umbilical cord showed a statistically significant decrease in expression of CD105 and CD34,
and increased CD90 expression. In terms of cell count, there were more cells surrounding the
vasculature and at either ends of the umbilical cord than in the centre. These findings warrant
further research into the functionality of MSC markers and the regions of the umbilical cord
with the highest MSC potencies.